- Email: [email protected]
Cytoplasmic progesterone receptors in the hypothalamus-preoptic area of the mouse: Effect of estrogen priming
0022-473 l/83 $3.00 + 0.00 Copynght IC 1983 Pergamon Press Ltd
J. s&mid Biochem. Vol. 19, No. 5, pp. 1571-1575, 1983 Printed in Great Britain. All rights reserved
CYTOPLASMIC PROGESTERONE RECEPTORS IN THE HYPOTHALAMUS-PREOPTIC AREA OF THE MOUSE: EFFECT OF ESTROGEN PRIMING* CHARLES E.
Department
of Physiology
and Biophysics,
RosELLtt$ and
CHARLES A. SNIPES$
Hahnemann University U.S.A. (Received
17 November
School of Medicine.
Philadelphia,
PA 19102,
1982)
Summary-A synthetic progestin, R5020, was used to identify cytoplasmic progestin receptors in the hypothalamuspreoptic area (HPOA) of ovariectomized mice. These high-affinity receptors exhibited an apparent dissociation constant of approx. 1 nM. The receptors were specific for progestins. [‘H]R5020 binding was inhibited by more than 50% with a SO-fold excess of either radioinert R5020 or progesterone. Su-Dihydroprogesterone inhibited binding to a lesser extent. 3a-Hydroxy-Sapregnane-20-one and cortisol did not compete for [‘H]R5020 binding. Administration of estradiol benzoate (10 kg), 48 h prior to death, resulted in a 54% increase in the HPOA progestin receptor concentration when compared to oil-injected controls. These data demonstrate that there are specific and saturable cytoplasmic progestin receptors in the mouse HPOA and that the concentration of these receptors is increased after estrogen treatment.
INTRODUCTION
Ovariectomized mice given estrogen injections display lordosis. This stereotypical female response to genital stimulation by a male is characterized by immobilization and concave arching of the back. When progesterone is given 48 h after estrogen, this behavioral response is enhanced [l]. In this regard. mice appear to resemble female guinea pigs, hams*Parts of this paper were presented as an abstract (No. 866) at the 63rd annual meeting of The Endocrine Society, Cincinnati, OH (1981). The work was supported in part by National Institutes of Health Biomedical Research Support Grant S-SO7 RR-05413. tPresent address: Reproductive Physiology. Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, OR 97006. *This work partially fulfilled the requirements for a Ph.D. degree for CER from Hahnemann University, Philadelphia. PA. DAddress reprint requests to Charles A. Snipes, Ph.D.. Department of Physiology and Biophysics, Hahnemann University, Philadelphia, PA 19102. The abbreviations and trivial steroid nomenclature used in this paper are: B. percentage of radioligand bound in the presence of competitor: B,,, percentage of radioligand bound in the absence of competitor; B,, number of specific
binding sites; cortisol, 1I/j’,17,21-trihydroxy-4-pregnene3,20-dione; 5a-dihydroprogesterone (Su-DHP), 5upregnane-3.20-dione; EDTA. ethylenediaminetetraacetic acid; HPOA. hypothalamus-preoptic area: K,,, apparent dissociation constant; 3a-0 1, 3a-hydroxy-5a-pregnan-20one; progesterone, 4-pregnene-3,20-dione; R5020, 17a.2 ldimethyl-19-norpregna-4.9-diene-3.20-dione; RBA, relative binding affinity; TED buffer, buffer comprising tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid, and dithiothreitol; Tris, tris(hydroxymethyl)aminomethane.
ters, and rats [2. 3,4]. It is generally accepted that in these species progesterone works synergistically with estrogen to facilitate the hormonal induction of sexual receptivity. Several lines of evidence suggest that progesterone acts through an intracellular receptor mechanism within neurons of the central nervous system to affect sexual behavior. Binding assays, involving both the potent synthetic progestin [“HI-17a.21-dimethyl-1% norpregna-4.9-diene-3.20-dione ([“H]R5020)* and [“HIprogesterone, have established the presence of cytoplasmic progesterone receptors in the brains of rats, guinea pigs, hamsters, hens and nonhuman primates [S-17]. The principal features of brain progestin receptors appear similar in all species thus far studied. The binding component is saturable and exhibits a high affinity for both R5020 and progesterone, with an apparent dissociation constant (Kn) in the nanomolar range. Sucrose density gradient centrifugation of the cytoplasmic hormone-receptor complex displays a characteristic sedimentation of 67s in low-ionic-strength buffers and 4.5s in highionic-strength buffers. Brain cytosol progestin receptors are highly specific for synthetic and naturally occurring progestins. Glucocorticoids and testosterone have practically no affinity for the receptors, and estradiol exhibits a low affinity. Competition studies have shown that So-dihydroprogesterone (Sa-DHP) has the highest affinity of any naturally occurring progesterone metabolite [S, 7, 10. 12). Although cytoplasmic progestin receptors are widely distributed throughout rat and guinea pig brains, some degree of tissue specificity can be inferred from the fact that estrogen pretreatment
1571
Cti.mtxs
1512
E. ROSEILI and CHARLES A. SNIPES
appreciably increases progestin receptors only in the hypothalamus and preoptic area [5, 121. These brain regions are crucially involved in the display of estrous behavior. Intracranial implants of progesterone in the hypothalamus of estrogen-primed rats facilitate the display of female sexual behavior [l&20]. The best evidence that a receptor mechanism is involved in progesterone-activated events is derived from the correlation, in terms of time course and estrogen dose. between the facilitation of lordosis and the induction of progesterone receptors [S. 13. 21, 22). The purpose of our study was to establish the presence of cytoplasmic progestin receptors in the mouse hypothalamus-preoptic area (HPOA) and to determine whether treatment with a behaviorally effective regimen of estradiol benzoate (231 affects the number or affinity of these receptors. We report the presence. in the mouse HPOA, of specitic and saturable cytoplasmic progestin receptors that are induced by prior treatment with estradiol benzoate.
MATERIALS AND METHODS
Steroids Radioinert R5020 and [ 17a-methyl-“H]R5020 (87 Ciimmol) were purchased from the New England Nuclear Corporation (Boston, MA). Radiochemical purity was assured by chromatography on thin layer plates (Bakerflex IB2-F) in a system of cyclohexaneether (1: 1). and the steroid was stored in absolute ethanol at +YC. The radioinert progesterone and SC+DHP were purchased from Mann Research (Division of Becton Dickinson, Rutherford, NJ). All other unlabeled steroids were obtained from the Sigma Chemical Company (St. Louis. MO). Animals Adult female CD-l mice, obtained from the Charles River Breeding Laboratories (Wilmington, MA), were maintained on a 12-h reversed light-dark cycle (dark: 1200-2400 h). Purina Lab Chow@ and water were available ad libitum. All mice were ovariectomized at 46 days of age, and 2 weeks later (day 60) were given subcutaneous injections: either 0.1 ml of peanut oil or 10 kg of 17B-estradiol benzoate in 0.1 ml of peanut oil. Groups of 10 mice were killed by decapitation 48 h after the injections. Each brain was removed onto a cold platform and bisected: the HPOA was removed. The HPOA was taken as the area extending from the posterior border of the mamillary body rostrally to a point 1 mm in front of the optic chiasm. The dorsal limit was taken as a line intersecting the ventral border of the thalamus. The tissue taken extended laterally to the edge of the tuber cinereum. The dissection included all the major hypothalamic and preoptic nuclei 1241. Tissuepreparation Brain tissues were homogenized in 16 vol of ice-cold TED buffer-10 mM tris(hydroxymethyl)-
aminomethane (Tris) HCI, 1.5 mM ethylenediaminetetraacetic acid (EDTA) and 0.25 mM dithiothreitol (pH 7.4)-with a ground-glass Kontes Dual1 apparatus. The homogenate was centrifuged for 1 h at 105,000 g with an SW SO. 1 rotor in a model L5-50 ultracentrifuge refrigerated to 1°C (Bet kman Instruments, Palo Alto, CA). The supernatant sytosol fraction was drawn from the pellet and used within 1 h. [‘H/R5020
binding assay
The procedures were modified versions of those described by Philibert and Raynaud (251. Cytosols (200 ~1) were incubated for 4 h at 0-4°C with 0.04-4.0 nM of [‘H]R5020 in the absence (total binding) or presence of a lOO-fold excess of radioinert RSO20 (nonspecific binding). All steroids were dissolved in ethanol (< 5% of the final volume) and TED buffer. and added to the incubates in a volume of 50 ~1 The reaction was terminated by the addition of 200 ~1 of a dextran-coated charcoal suspension (0.5% Norit A; Sigma Chemical Company) and 0.05% Dextran T70 (Pharmacia Fine Chemicals. Uppsala, Sweden) in 10 mM Tris-HCI and 1 .S mM EDTA (pH 7.4) and incubated with periodic agitation for I5 min at &4C. The charcoal was precipitated in a Sorval RCB-2 centrifuge (SM-24 rotor) at 1200 gfor IO min. Aliquots of bound steroid (200 ~1) were mixed with 10 ml of a xylene-based liquid scintillation cocktail (Aquasol. New England Nuclear Corporation). The samples were counted at 35% efficiency in a Packard Tri Carb scintillation counter or at 48% efficiency in a Beckman LS 8100 counter. Efficiencies were determined for each sample by the automatic external standard technique. All statistical comparisons were done with a two-tailed unpaired l-test [26]. Specific binding was calculated as the total binding minus the nonspecific binding. The cytosol protein content was measured according to Bradford‘s method [27]. and binding was expressed as femtomoles of [“H]R5020 bound per milligram of protein. Results were analyzed by the method of Scatchard
Pd. Ligand specificity ofl_‘H]R5020 binding sires Duplicate aliquots (200 ~1) of cytosol from the pooled hypothalami of 10 estrogen-treated ovariectomized mice were incubated for 4 h at (&-4”C with [‘H]R5020 in the absence or presence of various concentrations of unlabeled steroid competitors dissolved in 50 t.r.1of TED buffer. The final [‘H]RSO20 concentration was 1 x 1W” M, and the unlabeled steroid concentrations varied from lo-” M to lo-” M. Results, expressed as the percentage of radioligand bound in the presence of competitor (B) compared to that bound in the absence of competitor (B,,). were plotted against the log,,, of the competitor concentration. The relative binding affinity (RBA) of a competitor was estimated by calculating the ratio of unlabeled progesterone concentration which gives a
Mouse brain progestin receptors
E 0
600 0 0 500 0" g 400 k 300 10 P 200 _o
.
.
. s n 100 .u . 'cl__!IfC Y 2.0 :: Cytosol protZ concentration (mg/ml)
Fig. 1. Protein concentration and specific [“H]R5020binding. The hypothalamus-preoptic area (HPOA) cytosol was prepared from 10 adult ovariectomized CD-l mice that had been given a priming dose of estradiol benzoate (10 kg of EtB/O.l ml of peanut oil) 48 h prior to death. Duplicate ahquots (200 ~1) of several cytosol dilutions were incubated with 2.5 nM [3H]R5020 in the absence or presence of a lOO-fold excess of radioinert steroid. The reactions were incubated for 4 h at O-4 “C, then assayed by a dextrancoated charcoal technique to measure the amount of specific
binding.
50% reduction of [‘H]R5020 binding to the competitor concentration which gives a 50% reduction and multiplying by 100. The RBA of progesterone was taken to be 100 [29].
RESULTS
Time course of [-‘H]R5020 binding to cytosol
In the HPOA, specific binding of [3H]R5020 was initially rapid and reached completion within approximately 2 h. After equilibrium was reached, the amount of specifically bound [3H]R5020 remained constant for up to 16 h (data not shown). An incubation time of 4 h was therefore used in all subsequent experiments.
1573
and cortisol did not compete at the concentrations used. For unknown reasons, small concentrations or cortisol enhanced [“H]R5020 binding. Specific /3HJR5020 binding in HPOA cytosols from estrogen-primed and oil-injected ovariectomized mice
The following experiments were performed to determine whether the binding affinity or capacity of the progestin binding sites in the HPOA would be modified by a dose of estrogen normally used to prime females before administering a behaviorally facilitative dose of progesterone [23]. Therefore, groups of 10 adult ovariectomized CD-1 mice received either 10 kg of estradiol benzoate (subcutaneously) in 0.1 ml of peanut oil, or the oil vehicle alone, 48 h prior to death and receptor measurement. Saturation was achieved at a (“H]R5020 concentration of approximately l-3 nM verifying that progestin binding in HPOA cytosol was to a high-affinity saturable binder (data not shown). Scatchard plots of the data from a representative experiment. involving oil- and estrogen-primed mice, are shown in Fig. 3. In this experiment cytosols from the estrogen-treated mice exhibited a K,, of 1.05 x lo-” M and the number of specific binding sites (B,) was 72.8 fmolimg of cytosol protein. Binding in the oil-treated controls displayed a KC, of 1.23 x lo-” M and a B, of 47.4 fmolimg of cytosol protein. The mean Kd of seven independent determinations for estrogen-primed mice was 9.58 (+ 1.l SEM) X lo-“’ M, and the mean B, was 64.9 (k 3.0) fmolimg of cytosol protein. Five separate determinations for oil-treated controls gave a mean Kti of 1.27 (+ 0.2) X lo-’ M and an average B, of 42.2 (* 4.2) fmolimg of 120-
Effects of cytosolprotein concentration The amount of steroid binding as a function of the cytosol protein concentration is shown in Fig. 1.
Specific binding of [‘H]R5020 increased linearly with protein concentrations from 0.4 to 2.0 mg/ml. CytOsol protein concentrations between 1 and 2 mgiml were routinely used. L&and specificity of [3HIR5020 binding sites
The specificity of the high-affinity binding sites in HPOA cytosols was studied through examination of the ability of various unlabeled competitor concentrations (1O-y-1O-6 M) to inhibit the bindingof 1 nM [3H]R5020. The results of these experiments are illustrated in Fig. 2. Only the potent progestin R5020 and progesterone competed markedly with [3H]R5020 binding. The R5020 exhibited a relative binding affinity (RBA = 489) five times greater than that of progesterone (RBA = 100). The SWDHP had little effect, and exhibited an RBA of 5.6. The 3~01
I I I 10-G 10-g 10-e 10-7 Log,,competitor concentration (M)
Fig. 2. Specificity of cytoplasmic [‘H]R5020 binding sites in the hypothalamus-preoptic area (HPOA) of estrogenprimed female mice. Groups of 10 adult CD-l mice were primed, 2 weeks after ovariectomy, with 10 (*g of estradiol benzoate (subcutaneously) and killed 48 h later. Duplicate aliquots of HPOA cytosol(200 111)were incubated for 4 h at 0-4°C with 1 nM [‘H]R5020 in the absence of (B,) or presence (B) of increasing concentrations (l-1000 nM) of various competitors. Binding was measured by the dextran-coated charcoal technique. Abbreviations: 3~01, 3~ hydroxy-5a-pregnane-20-one; Sa-DHP, 5adihydroprogesterone: Prog, progesterone.
CHARLES
1514
“, 2
E.
ROSELLI
03 .
0”
.
E”
.
and
CHARLES
A.
SNIPES
behavioral potency of R5020 in rodents [32]. In the mouse brain Sa-DHP is the major metabolite of progesterone (331 and in CD-I mice is nearly ah effective as progesterone in facilitating estrous behavior 1231. However. this compound exhibits a low affinity for the HPOA progestin receptor (RBA = 5.6). Blaustein and Feder [5]. using a similar technique, reported an RBA of 20 for &DHP in the hypothalamus of the female guinea pig. Lee et (II. [lo]. however. found that a 50-fold excess of SU-DHP inhibits [3H]progesterone binding by 98% in hypothalamic cytosol of female rats. The latter report suggests that the competition between Scu-DHP and progesterone is greater than that indicated by binding studies involving [“H)R5020. This may be accounted for by the fact that R5020 has a greater affinity for the receptor and forms a more stable complex with the receptor than progesterone [34]. Nonetheless. our results indicate that Sa-DHP can bind to the progestin receptor, and this ability may account for its behavioral effects. Specific [‘H]R5020 binding in the HPOA cytosol is saturable and of limited capacity. Scatchard analysis indicates that the HPOAs from ovariectomized mice. given control injections of oil, contain a basal level of cytoplasmic progestin receptors. The concentration of receptor is enhanced by 54% 48 h after mice receive an injection of estradiol benzoate. This is ;I time during which the behavioral effects of progesterone are apparent in ovariectomized estrogen-primed mice [I]. Estrogen-inducible progestin receptors have also been found in hypothalamic and preoptic cytosols from both rats and guinea pigs and in midbrain cytosols from guinea pigs [5. 11. 131.These areas appear to be crucial to the neuroendocrine and behavioral effects of progesterone [ 1%Zlj. In fact. several groups (5. 13.20]. using both rats and guinea pigs. have shown that the time course for induction of hypothalamic progestin receptors closely parallels the interval in which progesterone facilitates the expression of sexual behavior. The demonstration that HPOA progestin receptor concentrations increase after estrogen priming, a necessry prerequicite for progesterone action. suggests that the central effects of progesterone may be mediated through a receptor mechanism that is essentially the same in all species thus far examined.
~~o.,~~.~ , $j
02
i\
IO Speclflcally
20
30 bound mg
40
50
['HIR5020 cytosol
60
70
(mol
x lOI
80 /
protein)
Fig. 3. Scatchard plots of cytoplasmic [‘H]R5020 binding in the hypothalamus-preoptic area (HPOA) of estrogen- and oil-injected female mice. Two weeks after ovariectomy. each adult CD-l mouse received a subcutaneous injection of either peanut oil (0.1 ml) or peanut oil plus estradiol benzoate (10 pg). Mice were killed 48 h after the injections. The HPOAs were excised and homogenized in buffer. Aliquots of the cytosol preparations (200 ~1) were incubated with 0.04 to 3.0 nM [- HJR5020 or [7H]R5020 in the presence of a lOO-fold excess of radioinert R5020 for 4 h at 0-4°C. Specific binding. assayed with the dextran-coated charcoal technique, was equivalent to the total binding minus the nonspecific binding.
cytosol protein. No statistically significant difference was found in Kd values for the two treatment groups. Estrogen treatment. however, significantly increased the concentration of cytoplasmic progestin receptors in the HPOA of ovariectomized mice (P < 0.005) when compared to oil-injected controls. These results indicate that specific and saturable high-affinity cytoplasmic progestin receptors are present in the mouse HPOA. and that the concentrations of these receptors are increased by a behaviorally effective priming dose of estradiol benzoate given 48 h prior to assay.
DISCUSSION
The study reported here clearly demonstrates the existence of an estrogen-inducible progestin receptor in the cytosol fraction of the mouse HPOA. The binding protein satisfies the major criteria expected of a specific progestin receptor as discussed by Clark and Peck [30]. The receptor displays a high affinity for [“H]R5020, a specific ligand for the progestin receptor [31]. The K,,, approx. 10e9 M, agrees with those reported for the progestin receptor in neural tissues from other species [5-8, 10-171. The competition data indicate that the assayed receptor is specific for progestins. A 50-fold excess or less of R5020 or progesterone inhibits [3H]R5020 binding by more than 50%. That the RBA is five times greater for R5020 (RBA = 489) than for progesterone (RBA = 100) agrees with the reported
REFERENCES
Ring J.R.: The estrogen-progesterone induction of sexual receptivity in the spayed female mouse. Ena’ocrinology 34 (1944) 269-275. Baling J.L. progesterone
and Blandau R.J.: The estrogeninduction of mating responses in the female rat. Endocrinology -25 (i939) 359-364.
spa&d Demosev E.W.. Hertz R. and Young W.C.: The expeiimental induction of oestrus (sexual receptivity) in the normal and ovariectomized guinea pig. Am. .I. Physiol. 116 (1936) 201-209.
Mouse
brain progestin
4. Frank A.H. and Fraps R.M.: Induction of estrus in the ovariectomized golden hamster. Endocrinology 37 (1945)
L.J. and Leavitt W.W.: Progestcrone receptor in the rat anterior pituitary: effect of estrogen priming and adrenalectomy. Steroids 31 ( 1978) 69-S 1. recep7. Kato .I. and Onouchi T.: Specific progesterone tors in the hypothalamus and anterior hypophvsis of the rat. Endochnology 101 (1977) 92C--928.. M.. Kamivoshi M. and Tanaka K.: A 8. Kawashima cytoplasmic progesterone receptor in hen pituitary and hypothalamic tissues. Endocrinology 102 (197X) 1207-
20.
21.
22.
23.
1213.
N.Y.
Chen T.J.
receptor
Acad.
and Allen T.C.: Regulation formation by estrogen action.
Sci. 286 (1977)
210-225.
10. Lee H.. Davies I.J. and Ryan K.J.: Progesterone receptor in the hypothalamic cytosol of female rats. Endocrinology
L.P. and Feder H.H.: Inhibition of lordosis behavior in ovariectomized guinea pigs by mesencephalit imolants of urogesterone. Brain Res. 70 (1974) 71-80.’ Powers J.B.: Facilitation of lordosis in ovariectomized rats by intracerebral progesterone implants. Brain Res. 48 (1972) 311-325. Moguilewsky M. and Raynaud J.-P.: The relevance of hypothalamic and hypophyseal progestin receptor regulation in the induction and inhibition of sexual behavior in the female rat. Endocrinologv 105 (1979) 516-522. Parsons B.. MacLusky N.H.. Krey L.. Pfaff D.W. and McEwen B.S.: The temporal relationship between estrogen-inducible gestin receptors in the female rat brain- and the time course of estrogen activation of mating behavior. Endocrinology 107 (IYSO) 774-779. Gorzalka B.B. and Whalen R.E.: Genetic regulation of hormone action: selective effects of progesterone and dihydroprogesterone (5a-pregnane-3,20-dione) on sexual receptivity in mice. Steroids 23 (1974) 499-506. Sidman R.L., Angevine J.B. Jr. and Pierce E.T.: Atlas of the Mouse Brain and Spinal Cord. Harvard University Press, Cambridge. MA (1971). Philibert D. and Raynaud J.-P.: Progesterone binding in the immature mouse and rat uterus. Steroids 22 1
169 (1979) 481-497. 6. Evans R.W.. Sholiton
Ann.
19. Morin
357-361.
5 Blaustein J.D. andFeder H.H.: Cytoplasmicprogestinreceptors in guinea pig brain: characteristics and relationship to the induction of sexual behavior. Brain Res.
9. Leavitt W.W., of progesterone
1575
receptors
104 (1979)
24.
791-800.
N.J. and McEwen B.S.: Oestrogen mod11. MacLusky ulates progestin receptor concentrations in some rat brain regions but not in others. Nature 274 (1978) 276278. 12. MncLuskv N.J. and McEwen B.S.: Progestin receptors in rat brain: distribution and properties of cytoplasmic progestin-binding sites. Endocrinology 106 (1980) 192202.
13. Moguilewsky M. and Raynaud J.-P.: Estrogensensitive progestin-binding sites in the female rat brain and pituitary. Brain Res. 164 (1979) 16.5-175. A.: Progestin receptors in the 14. Naess 0. and Attramadal pituitary and brain of the rat. J. steroid Biochem. 9 (1978) p. 879 (abstract No.325). IS. Seiki K. and Hattori M.: In vivouptake ofprogesterone by the hypothalamus and pituitary of the female ovariectomized rat and its relationship to cytoplasmic progesterone-binding protein. Endocr. jap. 20 (1973) 111-119. C. and Pirker R.: Progesterone 16. Spona J,, Bieglmayer receptor in the rat anterior pituitary. Transformation and nuclear translocation. FEBS Lett. 97 (1979) 269S. and Lim L.: Characterization of rat 17. Thrower hypothalamic progestin binding by spheroidal hydroxylapatite chromatography. Biochem. J. 186 (1980) 295300.
18. Feder H.H., Landau I.T.. Marrone B.L. and Walker W.A.: Interactions between estrogen and progesterone in neural tissues that mediate sexual behavior of guinea pigs. Psychoneuroendocrinologv 2 (1977) 337-347.
25.
(1973)
-
89-98.
B.J. : Starisrical Principles in Experimental DeMcGraw-Hill. New York (1962). 27. Bradford M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analvt. 26. Winer sign.
Biochem.
72 (1976)
248-258.
G.: The attractions of proteins for small 28. Scatchard molecules and ions. Ann. N.Y. Acad. Sci. 51 (1949) 660-672. 29. Ojasoo T. and Raynaud J.-P.: Unique steroid congeners for receptor studies. Cancer Res. 38 (1978) 4186 4198.
30. Clark J.H. and Peck E.J.: Female Sex Steroids: Receplors and Function. Springer-Verlag, Berlin (1979) p. 4. 31. Raynaud J.-P.: R.5020, a tag for the progestin receptor. In Progesterone Receptors in Normal and Neoplastic Tissues (Edited by W.L. McGuire. J.-P. Raynaud and E.E. Baulieu). Raven Press, New York (1977) p. 9. 32. Blaustein J.D. and Wade G.N. Progestin binding by brain and pituitary cell nuclei and female rat sexual behavior. Brain Res. 140 (1978) 36&367. of 33. Roselli C.E. and Snipes C.A.: In vitro metabolism progesterone to So-pregnanedione by brain in two strains of mice. Fedn. Proc. 39 (1980) p. 986 (abstract No.3780). 34. Moguilewsky M. and Raynaud J.-P.: Progestin binding sites in the rat hypothalamus, pituitary and uterus. Sferoids
30 (1977)
99-109.
J. s&mid Biochem. Vol. 19, No. 5, pp. 1571-1575, 1983 Printed in Great Britain. All rights reserved
CYTOPLASMIC PROGESTERONE RECEPTORS IN THE HYPOTHALAMUS-PREOPTIC AREA OF THE MOUSE: EFFECT OF ESTROGEN PRIMING* CHARLES E.
Department
of Physiology
and Biophysics,
RosELLtt$ and
CHARLES A. SNIPES$
Hahnemann University U.S.A. (Received
17 November
School of Medicine.
Philadelphia,
PA 19102,
1982)
Summary-A synthetic progestin, R5020, was used to identify cytoplasmic progestin receptors in the hypothalamuspreoptic area (HPOA) of ovariectomized mice. These high-affinity receptors exhibited an apparent dissociation constant of approx. 1 nM. The receptors were specific for progestins. [‘H]R5020 binding was inhibited by more than 50% with a SO-fold excess of either radioinert R5020 or progesterone. Su-Dihydroprogesterone inhibited binding to a lesser extent. 3a-Hydroxy-Sapregnane-20-one and cortisol did not compete for [‘H]R5020 binding. Administration of estradiol benzoate (10 kg), 48 h prior to death, resulted in a 54% increase in the HPOA progestin receptor concentration when compared to oil-injected controls. These data demonstrate that there are specific and saturable cytoplasmic progestin receptors in the mouse HPOA and that the concentration of these receptors is increased after estrogen treatment.
INTRODUCTION
Ovariectomized mice given estrogen injections display lordosis. This stereotypical female response to genital stimulation by a male is characterized by immobilization and concave arching of the back. When progesterone is given 48 h after estrogen, this behavioral response is enhanced [l]. In this regard. mice appear to resemble female guinea pigs, hams*Parts of this paper were presented as an abstract (No. 866) at the 63rd annual meeting of The Endocrine Society, Cincinnati, OH (1981). The work was supported in part by National Institutes of Health Biomedical Research Support Grant S-SO7 RR-05413. tPresent address: Reproductive Physiology. Oregon Regional Primate Research Center, 505 N.W. 185th Avenue, Beaverton, OR 97006. *This work partially fulfilled the requirements for a Ph.D. degree for CER from Hahnemann University, Philadelphia. PA. DAddress reprint requests to Charles A. Snipes, Ph.D.. Department of Physiology and Biophysics, Hahnemann University, Philadelphia, PA 19102. The abbreviations and trivial steroid nomenclature used in this paper are: B. percentage of radioligand bound in the presence of competitor: B,,, percentage of radioligand bound in the absence of competitor; B,, number of specific
binding sites; cortisol, 1I/j’,17,21-trihydroxy-4-pregnene3,20-dione; 5a-dihydroprogesterone (Su-DHP), 5upregnane-3.20-dione; EDTA. ethylenediaminetetraacetic acid; HPOA. hypothalamus-preoptic area: K,,, apparent dissociation constant; 3a-0 1, 3a-hydroxy-5a-pregnan-20one; progesterone, 4-pregnene-3,20-dione; R5020, 17a.2 ldimethyl-19-norpregna-4.9-diene-3.20-dione; RBA, relative binding affinity; TED buffer, buffer comprising tris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid, and dithiothreitol; Tris, tris(hydroxymethyl)aminomethane.
ters, and rats [2. 3,4]. It is generally accepted that in these species progesterone works synergistically with estrogen to facilitate the hormonal induction of sexual receptivity. Several lines of evidence suggest that progesterone acts through an intracellular receptor mechanism within neurons of the central nervous system to affect sexual behavior. Binding assays, involving both the potent synthetic progestin [“HI-17a.21-dimethyl-1% norpregna-4.9-diene-3.20-dione ([“H]R5020)* and [“HIprogesterone, have established the presence of cytoplasmic progesterone receptors in the brains of rats, guinea pigs, hamsters, hens and nonhuman primates [S-17]. The principal features of brain progestin receptors appear similar in all species thus far studied. The binding component is saturable and exhibits a high affinity for both R5020 and progesterone, with an apparent dissociation constant (Kn) in the nanomolar range. Sucrose density gradient centrifugation of the cytoplasmic hormone-receptor complex displays a characteristic sedimentation of 67s in low-ionic-strength buffers and 4.5s in highionic-strength buffers. Brain cytosol progestin receptors are highly specific for synthetic and naturally occurring progestins. Glucocorticoids and testosterone have practically no affinity for the receptors, and estradiol exhibits a low affinity. Competition studies have shown that So-dihydroprogesterone (Sa-DHP) has the highest affinity of any naturally occurring progesterone metabolite [S, 7, 10. 12). Although cytoplasmic progestin receptors are widely distributed throughout rat and guinea pig brains, some degree of tissue specificity can be inferred from the fact that estrogen pretreatment
1571
Cti.mtxs
1512
E. ROSEILI and CHARLES A. SNIPES
appreciably increases progestin receptors only in the hypothalamus and preoptic area [5, 121. These brain regions are crucially involved in the display of estrous behavior. Intracranial implants of progesterone in the hypothalamus of estrogen-primed rats facilitate the display of female sexual behavior [l&20]. The best evidence that a receptor mechanism is involved in progesterone-activated events is derived from the correlation, in terms of time course and estrogen dose. between the facilitation of lordosis and the induction of progesterone receptors [S. 13. 21, 22). The purpose of our study was to establish the presence of cytoplasmic progestin receptors in the mouse hypothalamus-preoptic area (HPOA) and to determine whether treatment with a behaviorally effective regimen of estradiol benzoate (231 affects the number or affinity of these receptors. We report the presence. in the mouse HPOA, of specitic and saturable cytoplasmic progestin receptors that are induced by prior treatment with estradiol benzoate.
MATERIALS AND METHODS
Steroids Radioinert R5020 and [ 17a-methyl-“H]R5020 (87 Ciimmol) were purchased from the New England Nuclear Corporation (Boston, MA). Radiochemical purity was assured by chromatography on thin layer plates (Bakerflex IB2-F) in a system of cyclohexaneether (1: 1). and the steroid was stored in absolute ethanol at +YC. The radioinert progesterone and SC+DHP were purchased from Mann Research (Division of Becton Dickinson, Rutherford, NJ). All other unlabeled steroids were obtained from the Sigma Chemical Company (St. Louis. MO). Animals Adult female CD-l mice, obtained from the Charles River Breeding Laboratories (Wilmington, MA), were maintained on a 12-h reversed light-dark cycle (dark: 1200-2400 h). Purina Lab Chow@ and water were available ad libitum. All mice were ovariectomized at 46 days of age, and 2 weeks later (day 60) were given subcutaneous injections: either 0.1 ml of peanut oil or 10 kg of 17B-estradiol benzoate in 0.1 ml of peanut oil. Groups of 10 mice were killed by decapitation 48 h after the injections. Each brain was removed onto a cold platform and bisected: the HPOA was removed. The HPOA was taken as the area extending from the posterior border of the mamillary body rostrally to a point 1 mm in front of the optic chiasm. The dorsal limit was taken as a line intersecting the ventral border of the thalamus. The tissue taken extended laterally to the edge of the tuber cinereum. The dissection included all the major hypothalamic and preoptic nuclei 1241. Tissuepreparation Brain tissues were homogenized in 16 vol of ice-cold TED buffer-10 mM tris(hydroxymethyl)-
aminomethane (Tris) HCI, 1.5 mM ethylenediaminetetraacetic acid (EDTA) and 0.25 mM dithiothreitol (pH 7.4)-with a ground-glass Kontes Dual1 apparatus. The homogenate was centrifuged for 1 h at 105,000 g with an SW SO. 1 rotor in a model L5-50 ultracentrifuge refrigerated to 1°C (Bet kman Instruments, Palo Alto, CA). The supernatant sytosol fraction was drawn from the pellet and used within 1 h. [‘H/R5020
binding assay
The procedures were modified versions of those described by Philibert and Raynaud (251. Cytosols (200 ~1) were incubated for 4 h at 0-4°C with 0.04-4.0 nM of [‘H]R5020 in the absence (total binding) or presence of a lOO-fold excess of radioinert RSO20 (nonspecific binding). All steroids were dissolved in ethanol (< 5% of the final volume) and TED buffer. and added to the incubates in a volume of 50 ~1 The reaction was terminated by the addition of 200 ~1 of a dextran-coated charcoal suspension (0.5% Norit A; Sigma Chemical Company) and 0.05% Dextran T70 (Pharmacia Fine Chemicals. Uppsala, Sweden) in 10 mM Tris-HCI and 1 .S mM EDTA (pH 7.4) and incubated with periodic agitation for I5 min at &4C. The charcoal was precipitated in a Sorval RCB-2 centrifuge (SM-24 rotor) at 1200 gfor IO min. Aliquots of bound steroid (200 ~1) were mixed with 10 ml of a xylene-based liquid scintillation cocktail (Aquasol. New England Nuclear Corporation). The samples were counted at 35% efficiency in a Packard Tri Carb scintillation counter or at 48% efficiency in a Beckman LS 8100 counter. Efficiencies were determined for each sample by the automatic external standard technique. All statistical comparisons were done with a two-tailed unpaired l-test [26]. Specific binding was calculated as the total binding minus the nonspecific binding. The cytosol protein content was measured according to Bradford‘s method [27]. and binding was expressed as femtomoles of [“H]R5020 bound per milligram of protein. Results were analyzed by the method of Scatchard
Pd. Ligand specificity ofl_‘H]R5020 binding sires Duplicate aliquots (200 ~1) of cytosol from the pooled hypothalami of 10 estrogen-treated ovariectomized mice were incubated for 4 h at (&-4”C with [‘H]R5020 in the absence or presence of various concentrations of unlabeled steroid competitors dissolved in 50 t.r.1of TED buffer. The final [‘H]RSO20 concentration was 1 x 1W” M, and the unlabeled steroid concentrations varied from lo-” M to lo-” M. Results, expressed as the percentage of radioligand bound in the presence of competitor (B) compared to that bound in the absence of competitor (B,,). were plotted against the log,,, of the competitor concentration. The relative binding affinity (RBA) of a competitor was estimated by calculating the ratio of unlabeled progesterone concentration which gives a
Mouse brain progestin receptors
E 0
600 0 0 500 0" g 400 k 300 10 P 200 _o
.
.
. s n 100 .u . 'cl__!IfC Y 2.0 :: Cytosol protZ concentration (mg/ml)
Fig. 1. Protein concentration and specific [“H]R5020binding. The hypothalamus-preoptic area (HPOA) cytosol was prepared from 10 adult ovariectomized CD-l mice that had been given a priming dose of estradiol benzoate (10 kg of EtB/O.l ml of peanut oil) 48 h prior to death. Duplicate ahquots (200 ~1) of several cytosol dilutions were incubated with 2.5 nM [3H]R5020 in the absence or presence of a lOO-fold excess of radioinert steroid. The reactions were incubated for 4 h at O-4 “C, then assayed by a dextrancoated charcoal technique to measure the amount of specific
binding.
50% reduction of [‘H]R5020 binding to the competitor concentration which gives a 50% reduction and multiplying by 100. The RBA of progesterone was taken to be 100 [29].
RESULTS
Time course of [-‘H]R5020 binding to cytosol
In the HPOA, specific binding of [3H]R5020 was initially rapid and reached completion within approximately 2 h. After equilibrium was reached, the amount of specifically bound [3H]R5020 remained constant for up to 16 h (data not shown). An incubation time of 4 h was therefore used in all subsequent experiments.
1573
and cortisol did not compete at the concentrations used. For unknown reasons, small concentrations or cortisol enhanced [“H]R5020 binding. Specific /3HJR5020 binding in HPOA cytosols from estrogen-primed and oil-injected ovariectomized mice
The following experiments were performed to determine whether the binding affinity or capacity of the progestin binding sites in the HPOA would be modified by a dose of estrogen normally used to prime females before administering a behaviorally facilitative dose of progesterone [23]. Therefore, groups of 10 adult ovariectomized CD-1 mice received either 10 kg of estradiol benzoate (subcutaneously) in 0.1 ml of peanut oil, or the oil vehicle alone, 48 h prior to death and receptor measurement. Saturation was achieved at a (“H]R5020 concentration of approximately l-3 nM verifying that progestin binding in HPOA cytosol was to a high-affinity saturable binder (data not shown). Scatchard plots of the data from a representative experiment. involving oil- and estrogen-primed mice, are shown in Fig. 3. In this experiment cytosols from the estrogen-treated mice exhibited a K,, of 1.05 x lo-” M and the number of specific binding sites (B,) was 72.8 fmolimg of cytosol protein. Binding in the oil-treated controls displayed a KC, of 1.23 x lo-” M and a B, of 47.4 fmolimg of cytosol protein. The mean Kd of seven independent determinations for estrogen-primed mice was 9.58 (+ 1.l SEM) X lo-“’ M, and the mean B, was 64.9 (k 3.0) fmolimg of cytosol protein. Five separate determinations for oil-treated controls gave a mean Kti of 1.27 (+ 0.2) X lo-’ M and an average B, of 42.2 (* 4.2) fmolimg of 120-
Effects of cytosolprotein concentration The amount of steroid binding as a function of the cytosol protein concentration is shown in Fig. 1.
Specific binding of [‘H]R5020 increased linearly with protein concentrations from 0.4 to 2.0 mg/ml. CytOsol protein concentrations between 1 and 2 mgiml were routinely used. L&and specificity of [3HIR5020 binding sites
The specificity of the high-affinity binding sites in HPOA cytosols was studied through examination of the ability of various unlabeled competitor concentrations (1O-y-1O-6 M) to inhibit the bindingof 1 nM [3H]R5020. The results of these experiments are illustrated in Fig. 2. Only the potent progestin R5020 and progesterone competed markedly with [3H]R5020 binding. The R5020 exhibited a relative binding affinity (RBA = 489) five times greater than that of progesterone (RBA = 100). The SWDHP had little effect, and exhibited an RBA of 5.6. The 3~01
I I I 10-G 10-g 10-e 10-7 Log,,competitor concentration (M)
Fig. 2. Specificity of cytoplasmic [‘H]R5020 binding sites in the hypothalamus-preoptic area (HPOA) of estrogenprimed female mice. Groups of 10 adult CD-l mice were primed, 2 weeks after ovariectomy, with 10 (*g of estradiol benzoate (subcutaneously) and killed 48 h later. Duplicate aliquots of HPOA cytosol(200 111)were incubated for 4 h at 0-4°C with 1 nM [‘H]R5020 in the absence of (B,) or presence (B) of increasing concentrations (l-1000 nM) of various competitors. Binding was measured by the dextran-coated charcoal technique. Abbreviations: 3~01, 3~ hydroxy-5a-pregnane-20-one; Sa-DHP, 5adihydroprogesterone: Prog, progesterone.
CHARLES
1514
“, 2
E.
ROSELLI
03 .
0”
.
E”
.
and
CHARLES
A.
SNIPES
behavioral potency of R5020 in rodents [32]. In the mouse brain Sa-DHP is the major metabolite of progesterone (331 and in CD-I mice is nearly ah effective as progesterone in facilitating estrous behavior 1231. However. this compound exhibits a low affinity for the HPOA progestin receptor (RBA = 5.6). Blaustein and Feder [5]. using a similar technique, reported an RBA of 20 for &DHP in the hypothalamus of the female guinea pig. Lee et (II. [lo]. however. found that a 50-fold excess of SU-DHP inhibits [3H]progesterone binding by 98% in hypothalamic cytosol of female rats. The latter report suggests that the competition between Scu-DHP and progesterone is greater than that indicated by binding studies involving [“H)R5020. This may be accounted for by the fact that R5020 has a greater affinity for the receptor and forms a more stable complex with the receptor than progesterone [34]. Nonetheless. our results indicate that Sa-DHP can bind to the progestin receptor, and this ability may account for its behavioral effects. Specific [‘H]R5020 binding in the HPOA cytosol is saturable and of limited capacity. Scatchard analysis indicates that the HPOAs from ovariectomized mice. given control injections of oil, contain a basal level of cytoplasmic progestin receptors. The concentration of receptor is enhanced by 54% 48 h after mice receive an injection of estradiol benzoate. This is ;I time during which the behavioral effects of progesterone are apparent in ovariectomized estrogen-primed mice [I]. Estrogen-inducible progestin receptors have also been found in hypothalamic and preoptic cytosols from both rats and guinea pigs and in midbrain cytosols from guinea pigs [5. 11. 131.These areas appear to be crucial to the neuroendocrine and behavioral effects of progesterone [ 1%Zlj. In fact. several groups (5. 13.20]. using both rats and guinea pigs. have shown that the time course for induction of hypothalamic progestin receptors closely parallels the interval in which progesterone facilitates the expression of sexual behavior. The demonstration that HPOA progestin receptor concentrations increase after estrogen priming, a necessry prerequicite for progesterone action. suggests that the central effects of progesterone may be mediated through a receptor mechanism that is essentially the same in all species thus far examined.
~~o.,~~.~ , $j
02
i\
IO Speclflcally
20
30 bound mg
40
50
['HIR5020 cytosol
60
70
(mol
x lOI
80 /
protein)
Fig. 3. Scatchard plots of cytoplasmic [‘H]R5020 binding in the hypothalamus-preoptic area (HPOA) of estrogen- and oil-injected female mice. Two weeks after ovariectomy. each adult CD-l mouse received a subcutaneous injection of either peanut oil (0.1 ml) or peanut oil plus estradiol benzoate (10 pg). Mice were killed 48 h after the injections. The HPOAs were excised and homogenized in buffer. Aliquots of the cytosol preparations (200 ~1) were incubated with 0.04 to 3.0 nM [- HJR5020 or [7H]R5020 in the presence of a lOO-fold excess of radioinert R5020 for 4 h at 0-4°C. Specific binding. assayed with the dextran-coated charcoal technique, was equivalent to the total binding minus the nonspecific binding.
cytosol protein. No statistically significant difference was found in Kd values for the two treatment groups. Estrogen treatment. however, significantly increased the concentration of cytoplasmic progestin receptors in the HPOA of ovariectomized mice (P < 0.005) when compared to oil-injected controls. These results indicate that specific and saturable high-affinity cytoplasmic progestin receptors are present in the mouse HPOA. and that the concentrations of these receptors are increased by a behaviorally effective priming dose of estradiol benzoate given 48 h prior to assay.
DISCUSSION
The study reported here clearly demonstrates the existence of an estrogen-inducible progestin receptor in the cytosol fraction of the mouse HPOA. The binding protein satisfies the major criteria expected of a specific progestin receptor as discussed by Clark and Peck [30]. The receptor displays a high affinity for [“H]R5020, a specific ligand for the progestin receptor [31]. The K,,, approx. 10e9 M, agrees with those reported for the progestin receptor in neural tissues from other species [5-8, 10-171. The competition data indicate that the assayed receptor is specific for progestins. A 50-fold excess or less of R5020 or progesterone inhibits [3H]R5020 binding by more than 50%. That the RBA is five times greater for R5020 (RBA = 489) than for progesterone (RBA = 100) agrees with the reported
REFERENCES
Ring J.R.: The estrogen-progesterone induction of sexual receptivity in the spayed female mouse. Ena’ocrinology 34 (1944) 269-275. Baling J.L. progesterone
and Blandau R.J.: The estrogeninduction of mating responses in the female rat. Endocrinology -25 (i939) 359-364.
spa&d Demosev E.W.. Hertz R. and Young W.C.: The expeiimental induction of oestrus (sexual receptivity) in the normal and ovariectomized guinea pig. Am. .I. Physiol. 116 (1936) 201-209.
Mouse
brain progestin
4. Frank A.H. and Fraps R.M.: Induction of estrus in the ovariectomized golden hamster. Endocrinology 37 (1945)
L.J. and Leavitt W.W.: Progestcrone receptor in the rat anterior pituitary: effect of estrogen priming and adrenalectomy. Steroids 31 ( 1978) 69-S 1. recep7. Kato .I. and Onouchi T.: Specific progesterone tors in the hypothalamus and anterior hypophvsis of the rat. Endochnology 101 (1977) 92C--928.. M.. Kamivoshi M. and Tanaka K.: A 8. Kawashima cytoplasmic progesterone receptor in hen pituitary and hypothalamic tissues. Endocrinology 102 (197X) 1207-
20.
21.
22.
23.
1213.
N.Y.
Chen T.J.
receptor
Acad.
and Allen T.C.: Regulation formation by estrogen action.
Sci. 286 (1977)
210-225.
10. Lee H.. Davies I.J. and Ryan K.J.: Progesterone receptor in the hypothalamic cytosol of female rats. Endocrinology
L.P. and Feder H.H.: Inhibition of lordosis behavior in ovariectomized guinea pigs by mesencephalit imolants of urogesterone. Brain Res. 70 (1974) 71-80.’ Powers J.B.: Facilitation of lordosis in ovariectomized rats by intracerebral progesterone implants. Brain Res. 48 (1972) 311-325. Moguilewsky M. and Raynaud J.-P.: The relevance of hypothalamic and hypophyseal progestin receptor regulation in the induction and inhibition of sexual behavior in the female rat. Endocrinologv 105 (1979) 516-522. Parsons B.. MacLusky N.H.. Krey L.. Pfaff D.W. and McEwen B.S.: The temporal relationship between estrogen-inducible gestin receptors in the female rat brain- and the time course of estrogen activation of mating behavior. Endocrinology 107 (IYSO) 774-779. Gorzalka B.B. and Whalen R.E.: Genetic regulation of hormone action: selective effects of progesterone and dihydroprogesterone (5a-pregnane-3,20-dione) on sexual receptivity in mice. Steroids 23 (1974) 499-506. Sidman R.L., Angevine J.B. Jr. and Pierce E.T.: Atlas of the Mouse Brain and Spinal Cord. Harvard University Press, Cambridge. MA (1971). Philibert D. and Raynaud J.-P.: Progesterone binding in the immature mouse and rat uterus. Steroids 22 1
169 (1979) 481-497. 6. Evans R.W.. Sholiton
Ann.
19. Morin
357-361.
5 Blaustein J.D. andFeder H.H.: Cytoplasmicprogestinreceptors in guinea pig brain: characteristics and relationship to the induction of sexual behavior. Brain Res.
9. Leavitt W.W., of progesterone
1575
receptors
104 (1979)
24.
791-800.
N.J. and McEwen B.S.: Oestrogen mod11. MacLusky ulates progestin receptor concentrations in some rat brain regions but not in others. Nature 274 (1978) 276278. 12. MncLuskv N.J. and McEwen B.S.: Progestin receptors in rat brain: distribution and properties of cytoplasmic progestin-binding sites. Endocrinology 106 (1980) 192202.
13. Moguilewsky M. and Raynaud J.-P.: Estrogensensitive progestin-binding sites in the female rat brain and pituitary. Brain Res. 164 (1979) 16.5-175. A.: Progestin receptors in the 14. Naess 0. and Attramadal pituitary and brain of the rat. J. steroid Biochem. 9 (1978) p. 879 (abstract No.325). IS. Seiki K. and Hattori M.: In vivouptake ofprogesterone by the hypothalamus and pituitary of the female ovariectomized rat and its relationship to cytoplasmic progesterone-binding protein. Endocr. jap. 20 (1973) 111-119. C. and Pirker R.: Progesterone 16. Spona J,, Bieglmayer receptor in the rat anterior pituitary. Transformation and nuclear translocation. FEBS Lett. 97 (1979) 269S. and Lim L.: Characterization of rat 17. Thrower hypothalamic progestin binding by spheroidal hydroxylapatite chromatography. Biochem. J. 186 (1980) 295300.
18. Feder H.H., Landau I.T.. Marrone B.L. and Walker W.A.: Interactions between estrogen and progesterone in neural tissues that mediate sexual behavior of guinea pigs. Psychoneuroendocrinologv 2 (1977) 337-347.
25.
(1973)
-
89-98.
B.J. : Starisrical Principles in Experimental DeMcGraw-Hill. New York (1962). 27. Bradford M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analvt. 26. Winer sign.
Biochem.
72 (1976)
248-258.
G.: The attractions of proteins for small 28. Scatchard molecules and ions. Ann. N.Y. Acad. Sci. 51 (1949) 660-672. 29. Ojasoo T. and Raynaud J.-P.: Unique steroid congeners for receptor studies. Cancer Res. 38 (1978) 4186 4198.
30. Clark J.H. and Peck E.J.: Female Sex Steroids: Receplors and Function. Springer-Verlag, Berlin (1979) p. 4. 31. Raynaud J.-P.: R.5020, a tag for the progestin receptor. In Progesterone Receptors in Normal and Neoplastic Tissues (Edited by W.L. McGuire. J.-P. Raynaud and E.E. Baulieu). Raven Press, New York (1977) p. 9. 32. Blaustein J.D. and Wade G.N. Progestin binding by brain and pituitary cell nuclei and female rat sexual behavior. Brain Res. 140 (1978) 36&367. of 33. Roselli C.E. and Snipes C.A.: In vitro metabolism progesterone to So-pregnanedione by brain in two strains of mice. Fedn. Proc. 39 (1980) p. 986 (abstract No.3780). 34. Moguilewsky M. and Raynaud J.-P.: Progestin binding sites in the rat hypothalamus, pituitary and uterus. Sferoids
30 (1977)
99-109.